PROJECT SUMMARY Primary human hepatocytes (PHHs) represent a scarce resource to build human liver models due to shortages of donor livers and thus these cells are not suitable for high-throughput drug screening. Induced pluripotent stem cell-derived human hepatocyte-like cells (iPSC-hHs) can address the aforementioned limitations of PHHs, allowing for the creation of genetically diverse donor panels to help elucidate inter-individual variations in drug response and disease progression. However, we and others have shown that iPSC-hHs need to be further differentiated towards the adult PHH phenotype. In vivo, a complex extracellular matrix (ECM) and a complex stromal compartment with multiple cell types modulate the hepatic phenotype. However, systematic evaluation of these cues on iPSC-hH functional maturation has not been evaluated to any considerable de- gree. It is well known that 3D microenvironments mimic human physiology better than 2D culture formats for many cell types (i.e. cancer, liver). Our main hypothesis is that a 3D microenvironment, which contains com- plex liver-inspired ECM coupled with key liver stromal cell types, will significantly differentiate iPSC-hHs to- wards the adult PHH phenotype. The challenge to testing this hypothesis using bulk hydrogels is that they are too labor/time intensive and costly to produce for high throughput exploration of optimal culture conditions. Thus, here we will utilize a high-throughput microtissue technology (>45,000 protein-based uniform microtis- sues per hour) to explore the aforementioned cues, which will allow us to have a large numbers of microtissues in each condition (>75) to obtain high statistical power in the results. In aim 1, we will investigate differentiated functions of iPSC-hHs in 3D microtissues of varying ECM compositions, while in aim 2, we will investigate dif- ferentiated functions of iPSC-hHs in co-culture with a complex liver-like stromal compartment in 3D microtis- sues with optimal ECM. Thus, our studies will create the first high-throughput 3D iPSC-hH / stromal co-culture platform with tunable ECM microenvironment, which can be used to investigate the chronic impacts of various stimuli (i.e. differentiation cues, chemicals, viruses, implantation sites for therapies) on liver functions.